In surface plasmon resonance (SPR) imaging, an optical imaging system is used to observe biomolecular binding events which have spatial structure. Generally, such a system includes a light source to illuminate a sample surface under conditions which produce SPR and a detector to image the light reflected from the sample surface.
FIG. 1A illustrates the SPR imaging principle. Light traveling through a high refractive index (RI) substrate 48 (e.g., BK7, n=1.51) reflects from a substrate surface, which is coated with a thin layer 51 of gold (50 nm). An aqueous sample (n=1.33), typically contained in a flowcell, contacts the opposite side of the gold. For certain wavelengths and angles of incident light, part of the incident energy will couple into a surface plasma wave traveling between the sample and the gold layer. The loss of this energy is observed as a decrease in reflectivity. Because the coupling conditions vary strongly with the refractive index of the sample, observations of reflectivity may be used as a sensitive measure of sample refractive index. Because the surface plasma wave is bound to the surface, the SPR phenomenon is only sensitive to the sample refractive index within the evanescent decay length, typically a few hundreds of nanometers. This surface sensitivity, combined with the fact that biomolecules such as proteins typically have refractive index much larger than water (n=1.6 typ.), allows the binding of biomolecules to the gold sensing surface to be detected as an increase in surface RI. To make an SPR imaging system for detection of specific substances, the gold surface is chemically functionalized (for instance by attaching antibodies to the surface) such that substances of interest will bind to the surface while other material will tend not to bind. Referring to FIG. 1A, a foundation layer 30 of biomolecules to which antibodies or other receptors (the “Y” molecules) are attached is illustrated adjacent to the gold layer 51.
FIG. 1B is a gray-scale plot that shows the tranverse magnetic (TM) reflectivity of the SPR sensing surface at various wavelengths, angles, and refractive indices. For a given refractive index (e.g., n=1.33), the plot shows the darkest region following a curve descending from approximately 600 nm at 76 degrees to 1000 nm at 64 degrees. When the refractive index increases to 1.36, for example, the dark region (the resonance position) moves higher in angle and wavelength. In SPR microscopy, both the angle and wavelength are fixed (i.e., a single x-y point is being examined on FIG. 1B), and brightness changes are observed due to changing refractive index. Thus, to sense refractive indices around 1.33, the wavelength and angle is set to some point on the dark curve for n=1.33.
Imaging the reflectivity of the sensing surface makes it possible to obtain a measurement of the refractive index at each point on the surface. The dashed curve shown in FIG. 2 illustrates the predicted variation of reflectivity with sample refractive index for monochromatic light (λ=670 nm) incident at a single angle (θ=69 degrees). The solid curve shows how the response is broadened when a range of wavelengths and angles (here 30 nm and 3 degrees) are included in the illumination.
As biomolecules bind to the surface, the surface refractive index (RI) will increase roughly proportional to the quantity of the substance that has bound. Observation of the RI over time will give a “binding curve,” such as those shown in FIG. 3, which reveals the quantity of bound material in real time. If the functionalization layer on the surface is patterned such that different regions of the surface tend to bind different substances, the changes in reflectivity which result as the surface is exposed to a sample may be analyzed to determine which of a number of substances are present in the sample, and in what concentration.
In optimizing an optical imaging system for use in observing SPR certain tradeoffs must be made between the following attributes, amongst others: refractive index resolution, spatial resolution and refractive index range. In particular, optimizing the detection limit of the system (in terms of molecular surface concentration) requires that the “signal” (i.e., the change in reflectivity which results from a binding event) be maximized, and that the “noise” (i.e., the uncertainty in the reflectivity measurement) be minimized, such that the signal-to-noise ratio (SNR) is maximized. With respect to spatial resolution, the optical imaging system ideally should be able to measure the variation of refractive index across the sensing surface with sufficient resolution to image any surface structure of interest. Finally, with respect to refractive index range, reflectivity increases linearly with RI for a range from approximately 1.325<n<1.335, as is shown in FIG. 2. To operate outside this range, the optical imaging system would typically be adjusted to change the incident angle and move the linear region to the desired location. Moreover, it is desirable to construct the system to require as little adjustment as possible.
In addition to optimizing the above attributes, it is desirable to produce an optical imaging system that is robust and inexpensive. Thus, it is also desirable to eliminate as many moving parts as possible and require little in the way of exotic optical components.
Therefore, there exists a need for an optical imaging system that is mechanically and optically simple, while also being capable of achieving high performance.